;;. Indi an Journal of Experimental Biology Vol. 43, March, 2005, pp. 254-258

In vivo effect of whole grain flour of finger millet (Eleusine coracana) and kodo millet (Paspalum scrobiculatum) on rat dermal wound healing

Prashant S Hegde 1, Anitha B2 & T S Chandra '*

'Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India 2Department of Biochemistry , Central Leather Research Insititute, Adyar, Chennai 600 020, Ind ia

Received 4 June 2004; revised 25 November 2004

Influence of finger millet and kodo millet on rat dermal wound healing was assessed by making a 4 cm2 (2 x 2 em) ex­cision wound on the shaven back of rats under ether anesthesia. Finger millet or kodo millet fl our (300 mg) as aqueous pas te was applied topicall y once daily for 16 days. The granulation tissue formed on day 4, 8 and 12 was used to estimate some biochemical parameters like protein , DNA, collagen and lipid perox ides. There was significant increase in protein and coll a­ge n contents and dec rease in lipid perox ides. Biophysical parameters like rate of contraction and number of days for epithe­liali zation were also studied. Rate of contraction was 88-90% in kodo millet and finger millet treated rats in compari son to 75% in untreated rats. The number of days for complete closure of wounds was lower for finger millet (13 days ) and kodo millet (14 days) treated rats in compari son to untreated (1 6 days) rats. The results implicate a possible therapeutical ro le fo r finger millet and kodo millet in accelerating the process of wound healing.

Keywords: Anti ox idant, Co llagen, Eleusine coracana, Finger millet, Free radicals, Kodo millet, Paspalum scrobiculatum, Wound hea ling,

IPC Code: Int C l7 A6 1 B

Wound healing and ti ssue repair are complex proc­esses that involve a seri es of biochemical and cellular reacti ons beginning with inflammation and followed by repair and remodelling of the injured ti ssue' . The formati on of cross links and development of tensile strength is mainly contributed by type I collagen in the wound matrix . However, type III collagen is the earlies t form of collagen detected in the wound2

. It fo rms scaffolding in the early wound healing process and subsequently the accumulation of type III colla­gen is overcome by the rapid accumulation of type I collagen (which provides mechanical integrity), until the normal ratio of type I to type Ill is re-es tablished3

.

The earl y type III coll agen has, in the healing process, important functions such as es tablishing initial wound structure, guiding inflammatory cells and fibroblasts into the wound site, providing a matrix for re­es tabli shment of blood supply . It also acts to regulate coll agen fibre diameter and organi zation.

Earlier studies on pos iti ve influence of Aloe vera , a tropical cactus, on the healing of wounds in diabetic rats indi cated that it may enhance the process by in-

' Correspondent author Telephone: 9 1-44-22578245; Fax : 9 1-44-22570305 Ema il : chandra@ iitm .ac.i n; tschandra @lycos.com

fluencing phases such as infl ammation, fibropl as ia, collagen synthes is and maturation, and wound con­traction. These effects may be due to the reported hy­poglycemic effects of the Aloe gd.

Similar studies on wounds treated with curcumin showed earlier re-epithelialization, improved neovas­cularization, increased migration of various ce ll s in­cluding dermal myofibrobl asts, fibroblasts, and macrophages into the wound bed, and a higher coll a­gen content5

. Plant products have been shown to have a good therapeutic potential as anti-inflammatory agents and promoters of wound healing, due to the presence of active alkaloids, terpenes and flavonoids6

-8

.

Finger millet and kodo mill et are rich source of polyphenols and exhibit signi ficant antioxidant activ­ity 9

· 10

• Owing to known ro le of antioxidants in wound healing and reports that phenolics like curcu­min exhibit anti-inflammatory activity5

, in the present study the finger millet and kodo mi llet fl our were analyzed for wound healing properties on topical ap­plication.

Materials and Methods Hydroxyproline, chloramine T , thiobarbituric acid,

1,1,3,3, tetra methoxy propane, bovine serum albumin

HEGDE et al.: EFFECT OF MILLET FLOUR ON DERMAL WOUND HEALING 255

and calf thymus DNA were obtained from Sigma­Aldrich Chemical Company, St Louis, USA. All other chemicals were of analytical grade commercially available.

Experimental set up - A standard full thickness wound (4 cm2) was created on the shaven back of male albino Wistar rats , 50-200 g, 3 months old, ob­tained from Tamilnadu Animal and Veterinary Sci­ence University, India under light anesthesia. Un­treated rats served as control (Group I); Group II rats were treated by topical application of 300 mg of aqueous paste of finger millet (FM) C0-13 flour and Group III rats were treated by topical application of 300 mg of aqueous paste of kodo millet (KM) Yam­ban 1 flour. In each group rats were taken in replica­tions (n=6). The granulation tissues formed were re­moved on day 4, 8 and 12 after wound creation and used to estimate the biochemical and biophysical parameters.

Extraction of total protein and DNA- The wet granulation ti ssue samples were extracted in Trichlo­roacetic acid (5 %)11. TCA (5 %; 10 ml) was added to the ti ssue samples (100 mg wet weight) and kept at 90° C for 30 min in a water bath to extract protein and DNA . The solution was centrifuged at 5000 rpm for I 0 minutes and the supernatant was used for the estimations.

Estimation of protein- Total protein was deter­mined by using Bovine Serum Albumin (BSA) as standard12. The total protein contents was expressed as mg/100 mg wet tissue.

Estimation of DNA- Tissue DNA content was determined by using calf thymus DNA as the stan­dard1 3. DNA content of tissue was expressed as mg/1 00 mg wet weight.

Estimation of total collagen- The total collagen content of the granulation tissue was determined by the estimation of hydroxyproline. The samples were washed with physiological saline, cut into small pieces, defatted with chloroform: methanol (2: I v/v) and lyophilized. Lyophilized tissue (5 mg) was digested with 6N HCl for 24 hr at 110° C. The amount of hydroxyproline was estimated as per Stegmann and Stalder14. Collagen content of tissue was expressed as mg/100 mg wet weight.

Estimation of lipid peroxides- Lipid peroxides (as malondialdehyde) of the tissue extracts were deter­mined as per Nagababu and Lakshmaiah 15 . The lipid peroxide content was expressed as n mole of MDA/100 mg wet weight.

Rate of contraction- The excision wounds were traced on a paper having a mm scale and the changes in wound size were measured planimetrically . Reduc­tion in wound (reflecting the rate of wound contrac­tion) was calculated as per cent of original wound size (4 cm2

) .

Period of epithelialization- The wounds were also inspected for complete epithelialization as indicated by shedding of eschar without any raw wound left behind. Days required for this sloughing was taken as the period of epithelialization.

Statistical analysis- All experiments were car­ried out with 6 rats in each group and results are ex­pressed as mean ± SD. Statistical analysis was done using Single way Anova and Newman-Keuls Multiple Comparison Test. P value of less than 0.05 was con­sidered significant.

Results and Discussion There was a significant increase in protein and

collagen contents on the day 4 and 8 of healing in FM and KM treated rats in comparison to control and a decrease by day 12 (Table 1). The levels of collagen and its types contribute significantly to the wound healing process. Collagen is the main protein in the extracellular matrix and plays an important role in the healing process even after the wound is closed 16. Though, the major function of collagen is to provide strength and integrity to the wound, it also plays a role in other functions such as homeostasis , re­epithelialization, cell-cell and cell-matrix interac­tions 17-20

.

Increase in total protein contents correlated well with increased collagen content in the granulation tissue in the millets treated group (Table l).The mil­lets could have stimulated collagen synthesis or in­creased the proliferation of fibroblasts synthesizing collagen or both. There was also an increase in DNA content on the day 4 and 8 of healing in FM and KM treated rats and a decrease on day 12 (Table I) .

There was a significant reduction in lipid peroxides on the day 4, 8, and 12 of healing in FM and KM treated rats in comparison to control (Table 1 ). The rate of wound contraction on the day 4, 8 and 12 was also higher in FM and KM treated rats (Table 1). The number of days for complete closure of wound de­creased significantly in the treated rats (Table 1 ).

The antioxidant and anti-inflammatory activities in extracts of leaves of Hyptis suaveoleni 1 and macro­fungus Phellinus rimosus (Berk) Pilat22 and

256 INDIAN J EXP BIOL, MARCH 2005

Table I - Total protein , col lagen, DNA, lipid peroxides content, rate of wound contraction of granulation tissues and period of ep i­thelialization in normal and experimental rats

[Values are mean ±SO of six animals in each group]

Biochemical parameters Group I Group II Group II I

Protein

a 3.57 ± 0.12 5.06 ± 0.11 u* 4.92 ± 0.09"' b 7.07 ± 0.08 8.63±0.!0"* 8.45 ± 0.08"* c 6.02 ± 0.09 7.70 ± 0.09"$ 7.59 ± 0.07"$

DNA

a 1.55 ± 0.07 1.89 ± 0.07 1.9 1 ± 0 08 b 5.39 ± 0.10 5.98 ± 0.08 5.75±0.16 c 4.26 ± 0.1 3 4.93±0.12 4.66 ± 0.12

Collagen

a 2.34 ± 0. 11 3.95 ± 0.12"' 3.78 ± 0.14"*

b 4.82 ± 0.16 7.04 ± 0.27"$ 6.56 ± 0. 1 0"$ c 3.78±0.16 5.35 ± 0.27"* 5.25 ± 0.17"'

Lipid peroxides

a 1357 ± 30.00 573 ± 13.00"" 648 ± IS .OO""h#

b 978 ± 15.00 374 ± 16.00"" 413 ± I O.OO"#b#

c 692 ± 24.00 20 I ± 7.00"# 302 ± I O.OO""h#

Rate of wound contraction

a 35 .00 ± 1.78 52.33 ± 0.81 a# 47.00 ± 0.89a#h#

b 50.00 ± 1.78 75 .66 ± 0.81 a# 71.33 ± 0.8l"#b# c 75 .16± 1.16 90.83 ± 0.98"# 88.16 ± 0.75a#b$

Period of epithelialization 15.83 ± 0.40 13. 50± 0.54"$ 14.00 ± 0.63"$

a, b, and c represent data on clay 4, 8, and 12 of healing.

Analysis clone wi th One-Way Anova and Newman-Keuls Multiple Comparison Test Comparisons are made between- "Group I and Group II, Group III ; bGroup II and Group III Values are significant at *P<O.OS, $?<0.01 , "P<O.OOI Values ex pressed for Protein, DNA and Collagen(mg/ 100 mg wet wt): Lipid

Peroxicles(n mole MDA/ I 00 mg wet wt); Rate of wound contraction(% of original wound size); Period of epitheli alization(clays)

supportive ro le of antioxidant enzymes in wound healing has been shown. Wound contraction in rats treated with whole plant extract of Leucas lavandu­laefolia Rees was better than application of a simple ointment23

. Similar reports on improved wound con­traction using methanolic extracts of leaf of Hyperi­cum patulum which contains steroids and fl avonoids is known24

. The healing effects of these extracts are attributab le to the synergistic ac tion of the phyto-h . I . h 21 c emtca s present m t em ·.

The mi ll ets used in the present study are rich source of phenolics9

· 10' 25

' 26 We have estab lished the

antioxidant activity of finger mi lle{ 27 and kodo mi l­let1 0' 27 phenolic extracts and showed that an intake of mi llets in diabetic rats improved wound healing28

.

Hence we attribute the reduction in lipid peroxides on day 4, 8, and 12 to the presence of rich antioxidant phenolics in these millets

Several other factors present in the millets that may have contributed to the accelerated wound healing in the treated rats include calcium and magnesium. They are reported to influence the process of wound heal ­ing29. Calcium has an established role in the normal homeostasis of mammalian skin and serves as a modulator in keratinocyte proliferation and differen­tiation. In wound repair, calcium is predominantly involved as Factor IV in the haemostatic phase, but it is expected to be required in epidermal cell migration and regeneration patterns in later stages of healing"). Finger millet is a rich source of calcium (344 mg/1 00 g) 31 . Hence, the accelerated wound healing in rats treated with finger millet could be due to presence of high calcium content.

During the early stages of wound tissue regenera­tion in rats, the methionine content of wound proteins is greater than the cystine content. Later, the content

HEGDE et al.: EFFECT OF MILLET FLOUR ON DERMAL WOUND HEALJNG 257

of cystine becomes much higher than that of methio­nine. This has been taken to mean that two different types of proteins are synthesized by the regenerating wound tissue. Hence, both the amino acids contribute significantly to increase the rate of regeneration of wound tissue32

. Finger and kodo millet contain sig­nificant amounts of methionine (210, 180 mg/ g N) and sulfur containing amino acid cysteine (140, 110 mg/ g N) Jt_

Conclusion This is the first study to show the potential useful­

ness of finger millet and kodo millet flour in topical application on the process of wound healing. Treat­ment with finger millet and kodo millet significantly increased levels of biochemical parameters like total protein, collagen and DNA content, and decreased the levels of lipid peroxides when compared to the con­trol. Biophysical parameters like increased rate of contraction and decrease in the number of days for epithelialization were observed. Treatment with finger millet showed better results in comparison to kodo millet. The nutraceutical s in these millets contributing to these beneficial properties need further study.

References Percival M, Nutritional support for connective ti ssue repair and wound healing, Clillical nwririon insights, NUT026 Rev.6/98, (Advanced Nutrition Publications, Inc) 1997, I.

2 Clore J N, Cohen , I K & Diegelmann R F, Quantitation of coll agen types I and 3 during wou nd healing in rat ski n, Proc Soc Exp Bioi Med, 161 (1979) 337.

3 Davidson J M & Benn S 1, Regulation of angiogenesis and wou nd repair, in Cellul ar and molecula·r pathogenesis, by A K Siren (Lippincott-Raven Publishers) 1996, 79.

4 Ch ithra P, Sajithlal G B & Chandrakasan G, Influence of Aloe vera on the healing of dermal wou nds in diabetic rats, J Ethllophamwcol, 59 ( I 998) 195.

5 Sidhu G S, Mani H, Gaddipati J P, Singh A K, Seth P, Banaudha K K, Patnaik G K & Maheshwari R K, Curcumin enhances wound healing in st reptozotocin induced diabetic rats and genetica ll y diabetic mice, Wound Repair Regellerat, 7 ( I 999) 362.

6 Porras- Reyes B H, Lewis W H, Roman J, Simchowitz L & Mustoe T A, Enhancement of wound healing by the alkaloid taspine defining mechanism of action, Proc Soc Exp Bioi Med, 203 (I 993) I 8.

7 Samuelsen A B, The traditional uses, chemical constituents and biological activities of Plantago major L, J Ethnophar· maca/, 71 (2000) l.

8 Mensah A Y, Sampson J, Houghton P J, Hylands P J, West­brook J, Dunn M, Hughes M A & Cherry G W, Effects of Buddleja globosa leaf and its constituents relevant to wound healin g, J Etlmophannacol, 77 (200 l) 219.

9 Sripriya G, Chandrasekharan K, Murthy V S & Chandra T S, ESR spectroscopic studies on free radical quenching action

of finger millet (Eleusine coracalla), Food Chem, 57 (1996) 537.

lO Prashant S H & Chandra T S, ESR spectroscopic studies re­veals higher free radical quenching potential in kodo millet (Paspalum scrobiculatwn) compared to other millets, Food Chem. (2004) Available Online 22"d October 2004.

ll Porat S, Roussa M & Shoshan S, Improvement of gliding function of flexor tendons by topically applied enriched col­lagen solution , J Bone Joint Surgery, 62 (1980) 208.

12 Lowry 0 H, Rosenbrough N J, Farr A L & Randall R T, Protein measurement with the Folin-phenol reagent , J Bioi Chem, 193(1951)265.

13 Burton K A, Study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of de­oxyribonucleic acid, Biochem J, 62 ( 1956) 315.

14 Stegmann H & Stalder K, Determination of hydroxyproline, Clin Chim Acta, 18 (1967) 267.

15 Nagababu E & Lakshmaiah N, Inhibitory effect of Eugenol on nonenzymatic lipid peroxidation in rat liver mitochondria, Biochem Pharmacal, 43 ( 1992) 2393.

16 Shoshan S, Wound healing, lilt Rev Connective Tissue Res, 9 (1981) I.

17 Raghow R, The role of ex tracellular matrix in postin­flammatory wound healing and fibrosi s, FASEB, 8 (I 994) 823.

18 Mutsaers S & Laurent G, Wound healing: From entropy to integrins, Biochemist, 17 ( 1995) 32.

i 9 Chithra P, Sajithlal G B & Chandrakasan G, Influence of Aloe vera on collagen turnover in healing of dermal wounds in rats, illdiall J Exp Bioi, 36 (1998) 896.

20 Suguna L, Sivakumar P & Chandrakasan G, Effects of Cell­tella asiatica extract on dermal wound healing in rats, illdiall J Exp Bioi, 34 (1996) 1208.

2 1 Shirwaikar A, Shenoy R, Udupa A L & Shetty S, Wound healing property of ethanolic extract of leaves of Hypris suaveolells with supportive role of antioxidant enzymes, In­dian J Exp Bioi, 41 (2003) 238.

22 Ajith T A & Janardhanan K K, Antioxidant and anti­inflammatory activities of methanol extract of Phellillus ri­mosus (Berk) Pilat, Indian J Exp Bioi, 39 (2001) 1166.

23 Saba K, Mukhetj ee P K, Das J, Pal M & Saba B P, Wound healing activity of Leucas lavalldulaefolia Rees, J Ethllo­phamwcol, 56 (1997) 139.

24 Mukhetjee P K, Verpoorte R & Suresh B, Evaluation of ill vivo wound healing activity of Hypericum patu/wn (Family: Hypericaceae) leaf extract on different wound model in rats, J Ethnopha nnacol, 70 (2000) 3 15.

25 Sripriya G, Usha A & Chandra T S, Changes in carbohy­drate, free amino acids, organ ic acids, phytate and HCL ex­tractability of minerals during germinati on and fermentation of finger millet (Eleusine coracana) , Food Chem, 58 ( 1997) 345.

26 Usha A & Chandra T S, Enzymatic treatment and use of starters for the nutrient enhancement in fermented flour of red and white varieties of finger millet (Eleusille coracalla) , J Agric Food Chem, 47 (1999) 2016.

27 Prashant S H, Gowri C & Chandra T S, Inhibition of col ­lage n glycation and crosslinking in vitro by methano li c extracts of finger millet (Eleusille coracana) and kodo millet (Paspalum scrobiculatum) , J Nutr Biochem, I 3 (2002) 517.

258 INDIAN J EXP BIOL, MARCH 2005

28 Rajasekaran N S, Nithya M, Rose C & Chandra T S, The ef­fect of finger millet feeding on the early responses during the process of wou nd healing in diabe tic ra ts, Biochim Biophys Acta, 1689 (2004) 190.

29 Grzesiak J J & Pierschbacher M D, Shifts in the concentra­tion of mag nesium and calcium in early porcine and rat wound fluids act ivate cell migratory response, J Clin In vest, 95 ( 1995) 227.

30 Lansdown A B, Calcium: A central regulator in wound healing in the sk in , Wound Repair Regenerat, 10 (2002) 27 1.

3 1 Gopalan C, Ramashastri B V & Balasubramaniam B V, Nu­tritive value of Indian foods (ICMR Offset Press, New Delhi) 1989,47.

32 Williamson M B & Fromm H J, The incorporation of sulfur amino acids into the proteins of regenerating wound ti ssue, J Bioi Chem, 2 12 ( 1955) 705.